Kawasaki Disease (KD) is a systemic inflammatory illness of childhood that causes coronary artery aneurysms in 25% of untreated patients and in 5% of those treated with intravenous gammaglobulin therapy. KD is the most common cause of acquired heart disease in children in developed nations. Clinical and epidemiologic features including seasonal epidemics and failure of antibiotic therapy strongly suggest a viral cause, but no known virus has been identified. My laboratory reported an antigen-driven IgA arterial immune response in acute KD, and synthetic versions of these antibodies identified intracytoplasmic inclusion bodies (ICI) in KD ciliated bronchial epithelium, in a subset of macrophages in lung, spleen, and lymph nodes, and in vascular tissue. Light and transmission electron microscopic analysis (TEM) of paraffin-embedded lung showed that the ICI are consistent with aggregates of viral proteins and RNA. Most recently, TEM of non- embedded bronchial tissue from three cases demonstrated virus-like particles (VLP) in close proximity to ICI. The VLPs were not ultrastructurally typical for any known virus. High-throughput (454) sequencing of cDNA from laser-capture microdissected bronchial epithelium from one of these cases (400,000 sequences) showed that interferon-stimulated genes were upregulated, consistent with viral infection. Stimulation of the alpha, beta interferon response in KD lung was subsequently confirmed using real-time PCR array analysis of KD and control lung samples. These findings led us to hypothesize that a new, previously unidentified RNA virus that enters through the lung is the causative agent of KD, and that viral sequences in KD tissues can be identified by high-throughput sequencing using a bioinformatics approach. This virus may have escaped detection to date because of limited nucleotide or amino acid homology with known viruses, and/or because an insufficient number of sequences from KD tissues have been examined. We have gathered a multidisciplinary team including KD infectious diseases and pathology experts, a virologist, an experienced geneticist, a bioinformatician, and a biostatistician, with the goal of identifying viral sequence(s) associated with KD. We propose the following specific aims for this exploratory grant: 1) Perform ultra high-throughput sequencing of cDNA amplified from KD tissues, 2) Use bioinformatics analysis to identify sequences with viral homology and without a match and perform assembly on sequences of interest to generate the longest contiguous sequences, and 3) Determine the presence of sequences of interest in additional KD and control tissues using real-time PCR analysis. The identification of ICI and VLP in KD tissues provides a unique opportunity to perform directed high-throughput sequencing to identify viral sequence(s) associated with KD, with the long-term goal of determining the etiologic agent of KD. Identification of the causative agent of KD would have profound implications for the field, enabling development of a diagnostic test, improved therapies, and ultimately, prevention of this potentially fatal illness of childhood.